Potent Inhibition of Telomerase by Small-Molecule Pentacyclic Acridines Capable of Interacting with G-Quadruplexes

Abstract

A novel pentacyclic acridine, 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4), has been identified as a potent inhibitor of telomerase in the cell-free telomeric repeat amplification protocol (TRAP). Modeling and biophysical studies suggest that RHPS4 inhibits telomerase through stabilization of four-stranded G-quadruplex structures formed by single-stranded telomeric DNA. In contrast to G-quadruplex interactive telomerase inhibitors described previously, RHPS4 inhibited telomerase at submicromolar levels (50% inhibition in the TRAP assay at 0.33 +/- 0.13 microM). Moreover, RHPS4 exhibited a wide differential between this potent inhibition of telomerase and acute cellular cytotoxicity (mean IC(50) value of 7.02 microM in 4-day growth inhibition assay). RHPS4, when added to 21NT breast cancer cells at nonacute cytotoxic concentrations (200 nM) every 3 to 4 days, induced a marked cessation in cell growth after 15 days. Similar effects were observed using another cell line possessing relatively short telomeres, A431 human vulval carcinoma cells, but not in a human ovarian carcinoma cell line (SKOV-3) possessing relatively long telomeres. In 21NT cells, growth cessation was accompanied by an increase in cells in the G(2)/M phase of the cell cycle, a reduction in cellular telomerase activity, and a lower expression of the hTERT gene. These effects occurred in the absence of a detectable reduction in telomere length as measured by slot blotting. RHPS4 also induced a cessation of growth of GM847 cells that maintain telomeres by a nontelomerase alternative mechanism for lengthening telomeres (ALT) after 15 days. RHPS4 represents a promising G-quadruplex interactive small molecule that is a potent cell-free inhibitor of human telomerase and induces growth inhibitory effects in human tumor cell lines after prolonged (2-week) exposure to nonacute cytotoxic drug concentrations.

Ascididemin and meridine stabilise G-quadruplexes and inhibit telomerase in vitro

“…The number of G4 ligands has grown rapidly over a few years: a range of molecules has been shown to inhibit telomerase through binding to its substrate [14,26,27,36,[44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59]. On the other hand, few natural products have been reported as G-quadruplex-mediated telomerase inhibitors, although one, telomestatin, is exceptionally potent with an IC50 of 5 nM against telomerase [25].…”

“…However, its calculated interaction energy remains modest compared to RHPS4 (-98.2 kcal/mol; data not shown). This positively-charged compound interacts with the human telomeric quadruplex, inhibits telomerase at submicromolar levels (IC 50 of 0.33 µM), induces growth inhibitory effects in human tumour cell lines as well as cell cycle alterations, apoptosis and telomere dysfunction [27,66]. This compound may also interact with the parallel-stranded DNA quadruplex d [TTAGGGT] 4 by stacking at the ends of the G-quadruplex [67].…”

“…Several reviews concerning telomerase inhibitors in general or quadruplex-based telomerase inhibitors have been published in the last few years [16][17][18][19][20][21][22][23]. The recent discovery of natural compounds such as cryptolepine [24] and telomestatin [25,26] or of synthetic pentacyclic acridines (such as RHPS4) [27] that interact with G-quartets led us to test the binding of ascididemin and meridine to G-quadruplexes.…”

“…Therefore, specific recognition of unusual DNA structures such as triplexes and quadruplexes by small molecules such as pyridoacridines should be possible. RHPS4 (center) [27,54,66,67]…”

G-Quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by loss of the 3′ telomeric overhang in cancer cells

“…Agents that stabilize G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalysed by telomerase, and thus are very attractive targets for drug design (Mergny and Helene, 1998). Small molecular ligands that selectively stabilize the telomeric G-quadruplex induce telomere shortening and replicative senescence (Han and Hurley, 2000;Gowan et al, 2001Gowan et al, , 2002Riou et al, 2002).…”